Project II, "Regulation of ENaC by Proteases and SGK1", has as its long-term objective the better understanding of the molecular mechanisms that link the rate of Na+ absorption by airway epithelia to physical conditions on the airway surface. Relevance to the mission of the agency is that the airway surface is the largest interface between the body and the outside environment, and the airway must continuously rid its surface of inhaled particulates. To do so, it has strongly conserved processes for regulating the height of the thin film of liquid on its surface. Excessive Na+ absorption mediated by airway epithelial Na+ channels (ENaC) contributes to lung disease in cystic fibrosis patients, but the mechanisms that normally restrain ENaC are poorly defined. To better understand how respiratory pathogens and/or inhaled chemicals could upset normal regulation in the airways, we propose to examine the regulation of ENaC by two pathways that may link the rate of Na+ absorption to stimuli present on the airway surface. The Project has thee aims. Aim 1: Extracellular proteolytic activity, as either soluble proteases, such as neutrophil elastase, or endogenous surface proteases, probably CAP1 (prostasin), stimulate near silent Po approx. 0.01) ENaC to become active (Po approx 0.5). Protease inhibitors, though essential for the model, are not addressed in this Project, which focuses on the molecular mechanism of proteolytic regulation. Aim 2: The role of ENaC surface density and turnover in airway health and disease is unknown. Aim 2 tests the effects of SGK1, ATP, and proteases on ENaC surface density and turnover. Aim 3 explores protease regulation of airway surface liquid volume in highly differentiated human airway cells in vitro and in a mouse model, in vivo. Our studies are focused on the effects of local signals on the open probability and surface density of ENaC, and how these modes of regulating Na+ transport satisfy the airway's requirement for tonic volume absorption, while preserving its ability to accelerate or slow the process as conditions on the airway surface require. Relevance to Public Health: This research is designed to study the physiologic processes responsible for clearing inhaled particles from the surface of the airways, so that the airways are able to deliver fresh air to the lungs' gas exchange surface.